Polyketides are structurally diverse natural products that include important therapeutic agents used as antibacterials (erythromycin), immunosuppressants (FK506), cholesterol lowering agents (lovastatin), and others (see Katz et al., 1993, Polyketide synthesis: prospects for hybrid antibiotics, Annu. Rev. Microbiol. 47: 875–912, incorporated herein by reference). Currently, there are about 7,000 identified polyketides, but this represents only a small fraction of what nature is capable of producing.
DNA sequencing of genes encoding several of the enzymes that produce type 1 modular polyketide synthases (PKSs) has revealed the remarkably logical organization of these multifunctional enzymes (see Cortes et al., 1990, An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea, Nature 348: 176–178; Donadio et al., 1991, Modular organization of genes required for complex polyketide biosynthesis, Science 252: 675–679; Schwecke et al., 1995, The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin, Proc. Natl. Acad. Sci. USA 92: 7839–7843; and August et al., 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699, Chem Biol 5: 69–79, each of which is incorporated herein by reference). The application of innovative combinatorial techniques to this genetic organization has prompted the generation of novel natural products, by adding, deleting, or exchanging domains or entire modules. See U.S. Pat. Nos. 5,672,491; 5,712,146; 5,830,750; 5,843,718; 5,962,290; and 6,022,731, each of which is incorporated herein by reference. It would be advantageous to have a practical combinatorial biosynthesis technology that could achieve and perhaps exceed the diversity of modular polyketide structures thus far revealed in nature.
The known modular PKSs have a linear organization of modules, each of which contains the activities needed for one cycle of polyketide chain elongation, as illustrated for 6-deoxyerythronolide B synthase (DEBS) in FIG. 1A. The minimal module contains a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP) that together catalyze a 2-carbon extension of the chain. The specificity of the AT for either malonyl or an alpha-alkyl malonyl CoA determines which 2-carbon extender is used, and thus the nature of the alkyl substituent at the alpha-carbon of the growing polyketide chain. After each 2-carbon unit condensation, the oxidation state of the beta-carbon is either retained as a ketone, or modified to a hydroxyl, methenyl, or methylene group by the presence a ketoreductase (KR), a KR+ a dehydratase (DH), or a KR+DH+ an enoyl reductase (ER), respectively. In effect, the AT specificity and the composition of catalytic domains within a module serve as a “code” for the structure of each 2-carbon unit. The order of the modules in a PKS specifies the sequence of the distinct 2-carbon units, and the number of modules determines the size of the polyketide chain.
The remarkable structural diversity of polyketides (see O'Hagan, The Polyketide Metabolites; Ellis Horwood, Chichester, 1991, incorporated herein by reference) is governed by the combinatorial possibilities of arranging modules containing the various catalytic domains, the sequence and number of modules, and the post-PKS “tailoring enzymes” that accompany the PKS genes. The direct correspondence between the catalytic domains of modules in a PKS and the structure of the resulting biosynthetic product allows rational modification of polyketide structure by genetic engineering.
Over the past several years, examples of modifying each of the elements that code for polyketide structure has been accomplished (see Kao et al., 1996, Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase, Biochemistry 35: 12363–12368; Liu et al., 1997, Biosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution, J. Am. Chem. Soc. 119: 10553–10554; McDaniel et al., 1997, Gain-of-function mutagenesis of a modular polyketide synthase, J. Am. Chem. Soc. 119: 4309–4310; Marsden et al., 1998, Engineering broader specificity into an antibiotic-producing polyketide synthase, Science 279: 199–202; and Jacobsen et al., 1997, Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase, Science 277: 367–369, each of which is incorporated herein by reference).
Recently, a combinatorial library of over 50 novel polyketides was prepared by systematic modification of DEBS, the PKS that produces the macrolide aglycone precursor of erythromycin (see U.S. patent application Ser. No. 09/429,349, filed Oct. 28, 1999; PCT patent application US99/24483, filed 20, Oct. 1999; and McDaniel et al., 1999, Multiple genetic modification of the erythromycin gene cluster to produce a library of novel “unnatural” natural products, Proc. Natl. Acad. Sci. USA 96: 1846–1851, each of which is incorporated herein by reference). With a single plasmid containing the eryAI, -AII and -AIII genes encoding the three DEBS subunits, ATs and beta-carbon processing domains were substituted by counterparts from the rapamycin PKS (see Schwecke et al., 1995, supra) that encode alternative substrate specificities and beta-carbon processing activities. The approach used was to develop single “mutations”, then sequentially combine the single mutations to produce multiple changes in the PKS. It was observed that when two or more single PKS mutants were functional, there was a high likelihood that combinations would also produce the expected polyketide. Although this strategy provided high assurance that the multiple mutants would be productive, the production of each polyketide required a separate engineering. Thus, if X mutants of eryAI, Y mutants of eryAII, and Z mutants at eryAIII were prepared, X+Y+Z separate experiments were required to produce that same number of polyketides. Clearly, the preparation of very large libraries by this approach is laborious.
Another strategy for preparing large numbers of polyketides is by random digestion-religation leading to “mutagenesis” of the domains or modules of a mixture of PKS genes, including the refinements embodied in the DNA shuffling method (see Patten et al., 1997, Applications of DNA shuffling to pharmaceuticals and vaccines, Curr. Op. Biotechnol. 8: 724–733, incorporated herein by reference). The expected low probability of assembling an active PKS by such an approach, however, would demand an extraordinary analytical effort (in the absence of a biological selection) to detect clones that produced polyketides within the much larger number of clones that are non-producers.
There remains a need for practical approaches to create large libraries of polyketides, non-ribosomal peptides, and mixed polyketides/non-ribosomal peptides.